Crystals of pyrroloquinolinequinone sodium salts

ABSTRACT

A crystal of pyrroloquinoline quinone disodium salt having peaks at 2θ of 9.1°, 10.3°, 13.8°, 17.7°, 18.3°, 24.0°, 27.4°, 31.2° and 39.5° (±0.2° for each) in powder X-ray diffractometry using Cu Kα radiation, or a crystal of pyrroloquinoline quinone trisodium salt having peaks at 2θ of 6.6°, 11.4°, 13.0°, 22.6°, 26.9°, 27.9°, 37.0°, 38.9° and 43.4° (±0.2° for each) in powder X-ray diffractometry using Cu Kα radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.13/383,472, filed Jan. 11, 2012, the entire contents of which areincorporated herein by reference. application Ser. No. 13/383,472 is a371 of International Application No. PCT/JP2010/059761, filed Jun. 9,2010, which is based upon and claims the benefits of priority toJapanese Application No. 2009-168087, filed Jul. 16, 2009.

TECHNICAL FIELD

The present invention relates to a novel crystal of a sodium salt ofpyrroloquinoline quinone and a method for producing it.

BACKGROUND ART

Since it has been proposed that pyrroloquinoline quinone (PQQ) may be anovel vitamin, it has drawn attention (see, for example, Non-patentDocument 1). PQQ exists in not only bacteria but also in molds andyeasts, which are eukaryotes, and plays important roles as a coenzyme.Further, PQQ has so far been known to have a number of bioactivitiessuch as the cell growth-promoting action, anti-cataract action, liverdisease-preventing/curing action, wound-healing action, anti-allergicaction, reverse transcriptase-inhibiting action, glyoxalase I-inhibitingaction, carcinostatic action and nerve fiber-regenerating action.

PQQ can be produced by an organic chemical synthetic method (Non-patentDocument 2), fermentation method (Patent Document 1) or the like.However, since PQQ obtained by these methods contains large amounts ofwater and impurities, a technology to obtain crystals of PQQ which arestable and highly pure has been demanded.

Crystals of a sodium salt of PQQ which were shown to be single crystalby X-ray crystallography have been obtained by evaporative concentrationof the disodium salt of PQQ in phosphate buffer (Non-patent Document 3).However, this method is not suitable for mass production. Further, in amethod by salting-out, since a salt used in a large amount, such asNaCl, is contaminated in the precipitated solids, there are drawbacks inthat an operation of removal of the existing salt is required and thatanalysis for securing stable quality is difficult because of the salt.

As a method for obtaining crystals of PQQ having no such drawbacks, amethod wherein a water-soluble organic solvent is added to causerecrystallization has been reported (Patent Document 2). However, thereis no information on the crystal form in this literature, and, when thepresent inventors carried out a confirmatory experiment forrecrystallization using ethanol, it was confirmed that the crystallinityof the disodium salt of PQQ was low and ethanol used as a solvent waslikely to remain even after drying, so that it was difficult to providecrystals having stable quality. Further, the ethanol recrystallizationmethod has a drawback in that the method requires a cooling apparatusfor precipitating solids, which increases the cost of the process.

Further, in terms of the trisodium salt of PQQ, there has been no reporton its crystal so far, unlike the disodium salt.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: JP 2751183 B

Patent Document 2: JP 7-113024 A

Non-Patent Documents

Non-patent Document 1: Nature, vol. 422, 24 April, 3003, p. 832

Non-patent Document 2: JACS, vol. 103, pp. 5599-5600 (1981)

Non-patent Document 3: JACS, vol. 111, pp. 6822-6828 (1989)

SUMMARY OF THE INVENTION

In consideration of the pharmaceutical value of PQQ, it is important toreproducibly obtain stable high-purity crystals having a definitecrystal form, which crystals can be stored for a long time withoutstrictly controlling storage conditions such as the temperature, light,humidity and oxygen level. Therefore, the present invention aims toprovide stable crystals of a sodium salt of PQQ and to provide a methodfor producing them.

The present inventors intensively studied to solve the above problems.As a result, the present inventors succeeded in obtaining novelhigh-purity crystals of sodium salts of PQQ, with which the aboveproblems can be solved, by studying the conditions of crystallization ofthe sodium salts of PQQ, thereby completing the present invention.

The present invention is as follows.

-   (1) A crystal of pyrroloquinoline quinone disodium salt having peaks    at 2θ of 9.1°, 10.3°, 13.8°, 17.7°, 18.3°, 24.0°, 27.4°, 31.2° and    39.5° (±0.2° for each) in powder X-ray diffractometry using Cu Kα    radiation.-   (2) A crystal of pyrroloquinoline quinone trisodium salt having    peaks at 2θ of 6.6°, 11.4°, 13.0°, 22.6°, 26.9°, 27.9°, 37.0°, 38.9°    and 43.4° (±0.2° for each) in powder X-ray diffractometry using Cu    Kα radiation.-   (3) A method for producing the crystal of pyrroloquinoline quinone    disodium salt according to (1), said method comprising preparing an    aqueous solution and/or suspension of pyrroloquinoline quinone    trisodium salt comprising a 10 to 90 (v/v) % water-soluble organic    solvent and adjusting pH of said aqueous solution and/or suspension    to within the range of 2 to 5 to crystallize pyrroloquinoline    quinone disodium salt.-   (4) The method according to (3), wherein said water-soluble organic    solvent is an alcohol.-   (5) The method according to (4), wherein said alcohol is ethanol.-   (6) The method according to any one of (3) to (5), wherein said    crystallization is carried out in a suspended state.-   (7) A method for producing the crystal of pyrroloquinoline quinone    trisodium salt according to (2), said method comprising adding a    water-soluble organic solvent to an aqueous solution and/or    suspension of pyrroloquinoline quinone trisodium salt such that a    concentration of 10 to 90 (v/v) % is attained while maintaining pH    of the obtained aqueous solution and/or suspension within the range    of 6 to 10, to crystallize pyrroloquinoline quinone trisodium salt.-   (8) The method according to (7), wherein said water-soluble organic    solvent is an alcohol.-   (9) The method according to (8), wherein said alcohol is ethanol.-   (10) A method for producing the crystal of pyrroloquinoline quinone    trisodium salt according to (2), said method comprising performing    salting-out while maintaining pH of an aqueous solution and/or    suspension of pyrroloquinoline quinone trisodium salt within the    range of 6 to 10, to crystallize pyrroloquinoline quinone trisodium    salt.-   (11) A functional food comprising the crystal of a sodium salt of    pyrroloquinoline quinone according to (1) or (2).-   (12) A pharmaceutical comprising the crystal of a sodium salt of    pyrroloquinoline quinone according to (1) or (2).

By the present invention, highly pure and stable crystals of a sodiumsalt of PQQ can be provided, and a method of their stable production canbe further provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a powder X-ray diffraction spectrum of thecrystals obtained in Example 1.

FIG. 2 is a diagram showing data from thermal analysis of the crystalsobtained in Example 1.

FIG. 3 is a diagram showing a powder X-ray diffraction spectrum of thecrystals obtained in Example 3.

FIG. 4 is a diagram showing a powder X-ray diffraction spectrum of thesolids obtained in Comparative Example 1 by the recrystallizationmethod.

FIG. 5 is a diagram showing a powder X-ray diffraction spectrum of thesolids obtained in Comparative Example 2 by crystallization in methanol.

FIG. 6 is a diagram showing a powder X-ray diffraction spectrum obtainedby the simulation in Comparative Example 3 using single-crystal data.

DESCRIPTION OF THE EMBODIMENTS

In the present invention, pyrroloquinoline quinone (PQQ) means thesubstance having the structure represented by Formula 1:

The crystal of the first invention of the present application(hereinafter referred to as Crystal 1) is a crystal of PQQ disodium salthaving peaks at the angle of reflection 2θ of 9.1°, 10.3°, 13.8°, 17.7°,18.3°, 24.0°, 27.4°, 31.2° and 39.5° (±0.2° for each) in powder X-raydiffractometry using Cu Kα radiation.

Measurement of the angle of diffraction 2θ by powder X-raydiffractometry can be carried out under, for example, the followingmeasurement conditions.

Apparatus: M18XCE, manufactured by MAC Science, Co. Ltd.

X-ray: Cu/40 kV tube voltage/100 mA tube current

Divergence slit: 1°

Scattering slit: 1°

Receiving slit: 0.3 mm

Scan speed: 4.000°/min.

Sampling width: 0.020°

The peaks can be monitored also with a common powder X-raydiffractometer equipped with a monochromator. Since the crystal formdefined in the present invention contains measurement errors, thedetermination can be carried out as long as rational identities for theangles of the peaks are observed.

In the cases of PQQ disodium salt obtained by the conventional ethanolrecrystallization method, such a large number of diffraction peaks didnot appear, so that the conventionally obtained solids are considered tohave low crystallinities.

Examples of the method for producing Crystal 1 include a method whereinPQQ trisodium salt is dissolved/suspended in water and a water-solubleorganic solvent is added to the resulting solution and/or suspensionsuch that a concentration of 10 to 90 (v/v) %, preferably 20 to 80 (v/v)% is attained, followed by adjusting pH to within the range of 2 to 5 tocause crystallization. Alternatively, the crystallization may be carriedout by dissolving and/or suspending PQQ trisodium salt in awater-soluble organic solvent and adding water to the resulting solutionand/or suspension such that the concentration of the water-solubleorganic solvent becomes 10 to 90 (v/v) %, preferably 20 to 80 (v/v) %,followed by adjusting pH to within the range of 2 to 5. Alternatively,the crystallization may be carried out by dissolving and/or suspendingPQQ trisodium salt in an aqueous medium containing a 10 to 90 (v/v) %,preferably 20 to 80 (v/v) % water-soluble organic solvent and adjustingpH to within the range of 2 to 5.

The PQQ trisodium salt to be used as a raw material can be produced byan organic chemical synthetic method (e.g., JACS, vol. 103, pp.5599-5600 (1981)), fermentation method (e.g., JP 1-218597 A and JP2692167 B) or the like. The PQQ trisodium salt to be used as a rawmaterial may be either crystalline or amorphous. The PQQ trisodium saltmay also contain impurities.

Particular examples of the water-soluble organic solvent which may beused include methanol, ethanol, n-propanol, isopropanol, ethyleneglycol, propylene glycol, methoxyethanol, diethylene glycol,methoxydiethylene glycol, glycerin, methoxypropanol, acetone, methylethyl ketone, acetonitrile, ethyl lactate and methyl hydroxyisobutyrate.Among these, alcohols are more preferred, ethanol is especiallypreferred.

The organic solvent is used as a poor solvent for decreasing thesolubility of PQQ, and the concentration of the organic solvent may beset appropriately within the above-described range depending on theinitial amount of PQQ trisodium salt.

In a common method for producing crystals, solids are dissolved in asolvent and crystals are then precipitated by adding a poor solvent tothe resulting solution.

However, in the present invention, the crystallization can be carriedout even in a suspended state in which solids exist. Being able to carryout the operation even in a suspended state is preferred since thevolume of the apparatus can be reduced and the amount of the wasteliquid to be discharged can be reduced.

In a particular method of operation to obtain Crystal 1, an aqueoussolution and/or suspension containing a raw material PQQ trisodium saltis first prepared. In this step, 0.5 to 80 g of PQQ trisodium salt per 1L of water is preferably added. Subsequently, a water-soluble organicsolvent is added to the aqueous solution and/or suspension to decreasethe solubility of PQQ trisodium salt. Precipitation of solids in thisstep is not problematic. Thereafter, an acid may be added to theresulting solution/suspension while measuring pH, thereby adjusting thepH to within the range of 2 to 5.

Alternatively, PQQ trisodium salt may be added to, and dissolved and/orsuspended in, a water-soluble organic solvent, followed by adding waterto the resulting solution and/or suspension and then adjusting pH towithin the range of 2 to 5, to cause crystallization. In this step, 0.5to 800 g of PQQ trisodium salt is preferably added per 1 L of the totalof the water-soluble organic solvent and the water added.

Alternatively, PQQ trisodium salt may be added to, and dissolved and/orsuspended in, an aqueous medium containing a water-soluble organicsolvent, followed by adjusting pH to within the range of 2 to 5, tocause crystallization. In this step, 0.5 to 800 g of PQQ trisodium saltis preferably added per 1 L of the aqueous medium.

The type of the acid to be added for the pH adjustment is notrestricted, and examples thereof include inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid,nitric acid and sulfuric acid; and organic acids such as formic acid,acetic acid, propionic acid, butyric acid, trichloroacetic acid,methanesulfonic acid and benzenesulfonic acid; among which hydrochloricacid is preferred.

When the pH became stable at a prescribed value within the range of pH 2to 5, precipitated crystals are separated by filtration, centrifugationor the like, and thereby Crystal 1 can be obtained.

When the water-soluble organic solvent is added to the aqueous solutionand/or suspension containing PQQ trisodium salt, the temperatures of theboth liquids are not restricted as long as the liquids are not frozen,and the temperatures are preferably −30° C. to 80° C. since a hightemperature decreases the energy efficiency. When the water-solubleorganic solvent is added and the acid is then added to adjust pH towithin the range of 2 to 5, the temperature of the solution orsuspension is not restricted as long as the liquid is not frozen, andthe temperature is preferably 5° C. to 80° C. since a high temperaturedecreases the energy efficiency and a low temperature decreases thecrystallization rate (speed). The liquid is preferably stirred asrequired and then left to stand. The stirring time may be, for example,5 minutes to 7 days. The standing time may be, for example, 5 minutes to15 days.

In the obtained solution, crystals of PQQ disodium salt (Crystal 1) areproduced. In the later-mentioned Example 2, the purity calculated byhigh performance liquid chromatography was 99.5%, so that extremelyhighly pure PQQ crystals can be obtained by this crystallization method.

Crystal 1 may be a hydrated material, and whether or not Crystal 1 ishydrated is not restricted as long as it has the above-described peaks.

The crystal of the second invention of the present application (Crystal2) is a crystal of PQQ trisodium salt having peaks at 2θ of 6.6°, 11.4°,13.0°, 22.6°, 26.9°, 27.9°, 37.0°, 38.9° and 43.4° (±0.2° for each) inpowder X-ray diffractometry using Cu Kα radiation. These peaks aredefined by powder X-ray diffractometry using the same measurement methodas in the case of the above Crystal 1.

Crystal 2 can be obtained by carrying out crystallization using anaqueous solution and/or suspension containing raw material PQQ trisodiumsalt as mentioned above at a pH within the range of 6 to 10.

The crystallization can be carried out by salting-out in the presence ofsodium ions or precipitation by addition of a water-soluble organicsolvent. Since, in the case of crystals of the trisodium salt, unlikethe case of crystals of the disodium salt, existence of a large excessof sodium ions is unlikely to make the analysis difficult, salting-outcan be employed without any problem. The crystals can be obtained byadjusting pH to within the range of 6 to 10 and performing salting-outby adding dietary salt, sodium sulfate, sodium nitrate, sodium phosphateor the like. The concentration of the salt is not restricted as long asthe added salt can be dissolved, and the concentration is preferably notless than 0.2 wt % and not more than 30 wt %. In cases where pH of theaqueous solution and/or suspension is within the range of 6 to 10, thepH adjustment is not necessary.

In cases where a water-soluble organic solvent is added, examplesthereof include methanol, ethanol, n-propanol, isopropanol, ethyleneglycol, propylene glycol, methoxyethanol, diethylene glycol,methoxydiethylene glycol, glycerin, methoxypropanol, acetone, methylethyl ketone, acetonitrile, ethyl lactate and methyl hydroxyisobutyrate.Among these, alcohols are more preferred, ethanol is especiallypreferred. The water-soluble organic solvent is preferably added suchthat a concentration of 10 to 90 (v/v) %, more preferably 20 to 80 (v/v)% is attained.

Crystal 2 may also be a hydrated material, and whether or not Crystal 2is hydrated is not restricted as long as it has the above-describedpeaks.

PQQ has many pharmacological effects such as the cell growth-promotingaction, anti-cataract action, liver disease-preventing/curing action,wound-healing action, anti-allergic action, reversetranscriptase-inhibiting action, glyoxalase I-inhibiting action,carcinostatic action and nerve fiber-regenerating action.

Therefore, Crystal 1 and Crystal 2 of PQQ of the present invention canbe used as effective ingredients of pharmaceuticals and functionalfoods. That is, the crystals can be provided in the forms of externalpreparations for the skin, injection solutions, oral preparations andsuppositories; and in the forms of foods and beverages taken regularly,dietary supplements, various hospital diets and the like. Examples ofadditives used for their preparation include liquids such as water;sugars such as fructose and glucose; oils such as peanut oil, soybeanoil and olive oil; and glycols such as polyethylene glycol andpolypropylene glycol. For solid preparations such as tablets, capsulesand granules, examples of vehicles include, but are not limited to,sugars such as lactose, sucrose and mannitol; examples of lubricantsinclude, but are not limited to, kaolin, talc and magnesium stearate;examples of disintegrators include, but are not limited to, starch andsodium alginate; examples of binders include, but are not limited to,polyvinyl alcohol, cellulose and gelatin; examples of surfactantsinclude, but are not limited to, fatty acid esters; and plasticizersinclude, but are not limited to, glycerin. Solubilizers, fillers and thelike may also be added as required.

Further, PQQ may be used either alone or in combination with othermaterials. Examples of materials which may be used in combinationinclude, but are not limited to, vitamins such as vitamin B complex,vitamin C and vitamin E; amino acids; astaxanthin; carotenoids such asα-carotene and β-carotene; ω3 fatty acids such as docosahexaenoic acidand eicosapentaenoic acid; and ω6 fatty acids such as arachidonic acid.

Since the PQQ crystals of the present invention (Crystal 1 and Crystal2) are highly pure and excellent in stability under preservation, thesecan be stored for a long time and preferably used as effectiveingredients of pharmaceuticals and functional foods.

The present invention will now be described in more detail by way ofExamples below, but the present invention is not restricted thereto.

EXAMPLES Reference Example 1

Raw material PQQ trisodium salt was obtained as follows.

According to Example 1 of JP 2692167 B, Hyphomicrobium methylovorumDSM1869 was cultured, and the obtained culture was centrifuged to removebacterial cells, to obtain the culture supernatant containing PQQ. Thisstrain can be obtained from DSM (Deutsche Sammlung von Mikroorganismen(German Collection of Microorganisms and Cell Cultures).

The culture supernatant was passed through a Sephadex G-10 column(manufactured by Pharmacia) to allow adsorption of PQQ, and elution wasperformed with an aqueous NaCl solution to obtain an aqueous PQQsolution at pH 7.5, followed by further adding NaCl to the solution to aconcentration of 60 g/L and cooling the resulting solution, therebyobtaining PQQ trisodium salt. The PQQ purity of the obtained PQQtrisodium salt was 99.0% as determined by UV absorption measured usinghigh performance liquid chromatography.

The sodium salt of PQQ was analyzed under the following conditions. ThePQQ concentration and purity were measured by high performance liquidchromatography under the conditions described below. Thereafter, theconcentration of Na contained in the same solution was measured bycation chromatography under the conditions described below. Further,based on the concentrations of PQQ and Na contained in the abovesolution, the ratio of the amounts of the substances PQQ and Nacontained in the sodium salt of PQQ was determined. In cases where thisratio of the amounts of the substances is 2.0±0.2, the salt is PQQdisodium salt, while in cases where the ratio is 3.0±0.2, the salt isPQQ trisodium salt.

(PQQ Analysis)

Apparatus: high performance liquid chromatography LC-20A, manufacturedby Shimadzu Corporation)

Column: YMC-Pack ODS-TMS (5 μm), 150×4.6 mm I.D.

Measuring temperature: 40° C.

Detection: Absorbance at 260 nm

Eluent: 100 mM CH₃COOH/100 mM CH₃COONH₄ (30/70, pH 5.1)

Elution rate: 1.5 mL/min.

(Na Analysis)

Pump: LC-6A, manufactured by Shimadzu Corporation

Column oven: HIC-6A, manufactured by Shimadzu Corporation

Measuring temperature: 40° C.

Detector: Conductivity meter CM-8000, manufactured by Tosoh Corporation

Column: Shodex, IC Y-521, manufactured by Showa Denko K. K.

Eluent: 4 mM HNO₃

Elution rate^(.) 1.0 mL/min.

Example 1 (Crystallization) Crystals of PQQ Disodium Salt

The PQQ trisodium salt prepared in Reference Example 1 was completelydissolved in ion-exchanged water, to prepare 500 g of a solutioncontaining 12 g/L PQQ. To this solution, 500 mL of ethanol was added. Atthis time, red solids were precipitated. While measuring pH using a pHmeter, hydrochloric acid was added dropwise to the resulting mixture toadjust pH to 3.6, followed by stirring the mixture at room temperaturefor 1 hour. Crystals were recovered by suction filtration and driedunder reduced pressure at room temperature for 16 hours. As a result,crystals of PQQ disodium salt were obtained in a yield of 95 mol %. Thepowder X-ray diffraction spectrum of the obtained crystals was measuredunder the conditions described below. The results are shown in FIG. 1.Peaks at 2θ of 9.1°, 10.3°, 13.8°, 17.7°, 18.3°, 24.0°, 27.4°, 31.2°,and 39.5°, ±0.2° for each, appeared in powder X-ray diffractometry usingCu Kα radiation.

Apparatus: M18XCE, manufactured by MAC Science, Co. Ltd.

X-ray: Cu/40 kV tube voltage/100 mA tube current

Divergence slit: 1°

Scattering slit: 1°

Receiving slit: 0.3 mm

Scan speed: 4.000°/min.

Sampling width: 0.02°

The obtained solids were crystalline.

Example 2 Production of Crystals of PQQ Disodium Salt

To the mixture of 500 mL of ion-exchanged water and 500 mL of ethanol,60 g of the solids of PQQ trisodium salt prepared in Reference Example1, which contain 20 g of PQQ, was added. At this time, the solids werenot completely dissolved. To the resulting mixture, hydrochloric acidwas added to adjust pH to 3.5. The addition of hydrochloric acid wascarried out by slowly adding hydrochloric acid dropwise to the mixturefor about 2 hours. After the pH became stable, the mixture was filtered,and crystals of PQQ disodium salt were thereby obtained in a yield of 99mol %. Although the purity of PQQ contained in the raw material PQQtrisodium salt was 99.0% as measured by liquid chromatography, thepurity of PQQ in the obtained PQQ disodium salt was 99.5%, whichcorresponded to 0.5% increase.

After drying the thus obtained crystals under reduced pressure, powderX-ray diffractometry was carried out in the same manner as in Example 1,and, as a result, the same results as in Example 1 were obtained

(Thermal Analysis)

The obtained solids were subjected to thermogravimetry (TG) anddifferential thermal analysis (DSC) using Thermo plus EVO TG8120manufactured by Rigaku Corporation. The temperature was increased underair flow at a rate of 10° C./minute.

The results are shown in FIG. 2. Except for occurrence of evaporation ofwater at about 100° C., there was no change until the temperatureincreased to 270° C., so that the substance was confirmed to be verystable.

The solids were analyzed by gas chromatography under the conditionsdescribed below, and, as a result, no residual ethanol was observed.

(Analysis of Residual Ethanol in Solids)

In 0.5 mL of distilled water, 50 mg of the obtained solids weresuspended, and ethanol contained in the supernatant obtained bycentrifugation was analyzed under the following conditions to determinethe ethanol content in the solids.

Apparatus: Gas chromatography GC-2014, manufactured by ShimadzuCorporation

Detection: FID, 210° C.

Carrier: High-purity nitrogen, 40 mL/min.

Example 3 Crystals of PQQ Trisodium Salt

The PQQ trisodium salt prepared in Reference Example 1 was completelydissolved in ion-exchanged water, to prepare 500 g of a solutioncontaining 12 g/L PQQ. Based on measurement with a pH meter, pH of theliquid was 7.0. To this liquid, 25 g of dietary salt was added. At thistime, red solids were precipitated. After stirring the resulting mixtureat room temperature for 1 hour, crystals were recovered by suctionfiltration and dried under reduced pressure at room temperature for 16hours. As a result, crystals of PQQ trisodium salt were obtained in ayield of 98 mol %. The powder X-ray diffraction spectrum of the obtainedcrystals was measured under the conditions described below. The resultsare shown in FIG. 3. Peaks at 2θ of 6.6°, 11.4°, 13.0°, 22.6°, 26.9°,27.9°, 37.0°, 38.9° and 43.4°, ±0.2° for each, appeared in powder X-raydiffractometry using Cu Kα radiation.

Apparatus: M18XCE, manufactured by MAC Science, Co. Ltd.

X-ray: Cu/40 kV tube voltage/100 mA tube current

Divergence slit: 1°

Scattering slit: 1°

Receiving slit: 0.3 mm

Scan speed: 4.000°/min.

Sampling width: 0.02°

The obtained solids were crystalline.

Example 4 Crystals of PQQ Trisodium Salt

The PQQ trisodium salt prepared in Reference Example 1 was completelydissolved in ion-exchanged water, to prepare 500 g of a solutioncontaining 12 g/L PQQ. Based on measurement with a pH meter, pH of theliquid was 7.0. To this liquid, 500 mL of ethanol was added. At thistime, red solids were precipitated. After stirring the resulting mixtureat room temperature for 1 hour, crystals were recovered by suctionfiltration and dried under reduced pressure at room temperature for 16hours. As a result, crystals of PQQ trisodium salt were obtained in ayield of 89 mol %. The X-ray diffraction spectrum of the obtained powderof crystals was measured under the same conditions as in Example 3.

As a result, peaks at the same positions as in Example 3 were observed.

In the same manner as in Example 2, thermal analysis and analysis of theresidual ethanol in the solids were carried out, and, as a result, thesolids were confirmed to have very high thermal stability, and noresidual ethanol was observed.

Comparative Example 1 Precipitation of Solids of PQQ Disodium Salt byEthanol Recrystallization

The PQQ trisodium salt prepared in Reference Example 1 was completelydissolved in ion-exchanged water, to prepare 800 g of a solutioncontaining 10 g/L PQQ. Hydrochloric acid was added to the solution toadjust the pH to 3.5, and 200 mL of ethanol was then added to thesolution. At this time, red solids were precipitated. After stirring theresulting mixture at room temperature for 5 hours, the mixture was leftto stand at 5° C. for 24 hours, to precipitate solids. The solids wererecovered by continuous centrifugation, and dried at 50° C. underreduced pressure.

The powder X-ray diffraction spectrum of the obtained solids wasmeasured under the same conditions as in Example 1. The results areshown in FIG. 4. Since the obtained solids hardly showed peaks exceptfor those in the low-angle side, these were confirmed to have lowcrystallinity.

The solids were analyzed by gas chromatography under the same conditionsas in Example 2, and, as a result, 0.02% residual ethanol was observedin the solids.

Further, in the same manner as in Example 2, thermal analysis wascarried out, and, as a result, reduction in the weight was observed fromabout 30° C., so that the solids were confirmed to be unstable.

Comparative Example 2 Precipitation of Solids of PQQ Disodium Salt byCrystallization in Methanol

The experiment of crystallization of solids of PQQ disodium saltdescribed in Patent Document 2 was reproduced and the obtained crystalswere compared with those of the present invention.

In 30 mL of water at about 60° C., 0.21 g of the crystals of PQQdisodium salt obtained in Example 1 were dissolved. To this solution, 50g of methanol was added. At the time of the mixture, the solution wasuniform. By leaving the solution to stand at 4° C. overnight, solidswere precipitated. The mixture was centrifuged and dried under reducedpressure, and the powder X-ray diffraction spectrum of the obtainedsolids was then measured under the same conditions as in Example 1. Theresults are shown in FIG. 5. Peaks at positions different from thoseobserved in Examples 1 and 2 in the present application were observed.

Further, in the same manner as in Example 2, thermal analysis andanalysis of the residual ethanol in the solids were carried out, and, asa result, the solids were confirmed to have low thermal stability, andresidual ethanol was observed.

Comparative Example 3 Conversion of Single Crystal Data to Powder X-rayData

For the PQQ disodium salt reported in JACS, vol. 111, pp. 6822-6828(1989), the data of X-ray crystallography of single crystals describedin this literature were used to simulate how peaks appear in powderX-ray diffractometry of these crystals.

Results of calculation using analysis software RIETAN2000 with WINDOWS(registered trademark) XP and a personal computer having a CPU of notless than 1 GHz are shown in FIG. 6.

As shown therein, there are peaks at positions different from those inthe cases of the crystals of the present invention.

The results from Examples 1 and 3 and Comparative Examples 1, 2 and 3are summarized below in Table 1.

TABLE 1 Exam- Comparative Exam- Comparative Comparative ple 1 Example 1ple 3 Example 2 Example 3 PQQ salt composition Tri- Disodium Disodiumsodium Disodium Disodium Crystal- salt salt salt salt salt lizationExam- Comparative Exam- Comparative Comparative method ple 1 Example 1ple 3 Example 2 Example 3 2θ 9.1 9.7 6.6 7.5 5.1 10.3 21.1 11.4 8.6 7.913.8 27.5 13.0 10.2 10.1 17.7 22.6 13.8 15.8 18.3 26.9 18.3 16.2 24.027.9 22.6 23.3 27.4 37.0 23.9 27.8 31.2 38.9 27.5 29.2 39.5 43.4

The values at 2θ were clearly different from each other, suggesting thatthe crystals have different crystal forms. It was shown that the crystalforms of the present invention are novel.

INDUSTRIAL APPLICABILITY

The PQQ crystals of the present invention are useful in the fields ofpharmaceuticals, functional foods and the like.

1. (canceled)
 2. A functional food, comprising: a crystal ofpyrroloquinoline quinone disodium salt having peaks at 2θ of 9.1°,10.3°, 13.8°, 17.7°, 18.3°, 24.0°, 27.4°, 31.2° and 39.5° (±0.2° foreach) in powder X-ray diffractometry using Cu Kα radiation.
 3. Thefunctional food of claim 2, wherein the crystal has a purity of 99.5% orhigher as assayed by high performance liquid chromatography.
 4. Thefunctional food of claim 2, further comprising: at least one additivewhich includes at least one of water, sugar, oil, and a glycol.
 5. Afunctional food, comprising: a crystal of pyrroloquinoline quinonetrisodium salt having peaks at 2θ of 6.6°, 11.4°, 13.0°, 22.6°, 26.9°,27.9°, 37.0°, 38.9° and 43.4° (±0.2° for each) in powder X-raydiffractometry using Cu Kα radiation.
 6. The functional food of claim 5,further comprising: at least one additive which includes at least one ofwater, sugar, oil, and a glycol.